The present invention relates in general to shuttle vectors for use with E. coli and Streptomyces and cloning of antibiotic genes using these vectors. In particular, the present invention relates to vectors pNJl and pAL7002 and to production of 2-norerythromycin antibiotics employing these vectors.
Cloning vectors, called E. coli-Streptomyces shuttle vectors, may be constructed which contain origins of replication and antibiotic resistance markers for both E. coli and in Streptomyces. Nakatsukasa et al., U.S. Pat. No. 4,513,085; Fayerman et al., U.K. Patent Application No. 2,107,716; Fayerman et al., U.K. Patent Application No. 2,107,717; and Hershberger et al., U.K. Patent Application No. 2,118,947. However, because Streptomyces strains are used to produce most of the clinically important antibiotics, it is desirable to obtain stable vectors which replicate and are selectable in E. coli, where recombinant manipulations are relatively easy to perform, and which replicate and are selectable in Streptomyces, wherein recombinantly-manipulated genes are expressed. Stability is particularly important inasmuch as some such shuttle vectors may be unstable, i.e. may be lost from their host cell along with the cloned gene they carry. See e.g. Matsushima et al., Bio/Technology, 4, 229-232 (1986).
Because cosmid vectors combine the opportunity to make use of the wide variety of features of plasmid vectors, including selection markers, and the large cloning capacity of phage vectors, they are particularly desirable. E. coli-Streptomyces cosmid shuttle vectors may be constructed to take advantage of these features. Matsushima et al., supra. Cosmid shuttle vectors which are stable with application of selection pressure are also desirable.
Erythromycin is a macrolide antibiotic product of a biosynthetic pathway in Streptomyces erythreus which is believed to have about thirty steps. Cosmid shuttle vectors carrying genes for antibiotic production may be used to explore biosynthesis of hybrid antibiotics. Matsushima et al., supra. Attempts to assay for the production of erythromycin may involve the use of S. erythreus strains having no detectable antibiotic activity which are blocked in the biosynthesis of erythromycin, such as eryA mutants which are blocked in the formation of 6-deoxyerythronolide B, an unmodified lactone. Matsushima et al., supra. Restoration of antibiotic function in such strains after transformation with a vector carrying genetic material to be tested for the production of erythromycin indicates antibiotic function is restored by the vector-borne genetic material. However, no suggestion has been made that such a technique might be employed to obtain hitherto unreported analogs of erythromycin such as norerythromycins.